Orthogonal translation of modified proteins featuring genetically encoded non-natural amino acids (nnAA) such as photocrosslinkers, photocaged groups, biophysical probes, posttranslational modifications, and proximity enhanced bioreactive groups has become an important tool for the study of biological processes and has achieved a long-standing goal of synthetic biology. A critical requirement for site-specific incorporation of nnAA into a custom protein is the ability to selectively catalyze aminoacylation of an orthogonal tRNA with the specified amino acid. Therefore, directed evolution of a new orthogonal aminoacyl-tRNA synthetase (aaRS) is required for each new nnAA that is added to the growing amino acid repertoire. While repeated cycles of positive and negative selection have allowed nonnatural aminoacylation activity to be evolved successfully, these efforts typically utilize only 3-5 total rounds of evolution due to the difficulty and time investment that is required to carry ot each round of traditional laboratory evolution. Unfortunately, these efforts result in evolved aaRS enzymes with relatively modest selectivity and greatly reduced activity compared to wild-type enzymes. This proposed work would test the hypothesis that selecting for new aaRS variants through massively increased total rounds of evolution will allow highly selective aaRSs to be evolved with wild-type-like activity. Phage-assisted continuous evolution (PACE) enables proteins to be evolved in the laboratory at a rate of up to ~40 rounds of evolution every 24 hours with minimal researcher intervention. In this proposed research, positive and negative selections using PACE will be used in an attempt to rapidly evolve enhanced orthogonal aaRSs capable of selective aminoacylation with desired nnAAs. Specific Aim 1 seeks to improve the selectivity of the p-nitrophenylalanyl-tRNA synthetase, which has previously been used to site-selectively incorporate a proximity probe into a designer protein and has also successfully been used as an immunogenic hapten in a protein. Specific Aim 2 will attempt to improve upon the inherently low activity of the pyrrolysyl-tRNA synthetase (PylRS), which has permitted genetic code expansion in cells and animals including mammals. Finally, Specific Aim 3 will seek to evolve a variant of the PylRS that is capable of selective aminoacylation with unprotected N6,N6-dimethyl-L-lysine in the presence of cellularly abundant L-lysine, which would have great utility in studies of posttranslational modifications such as histone methylation. If successful, this research will establish a new platform for rapidly evolving enhanced orthogonal aaRS enzymes to greatly expand their use in synthetic biology and therapeutic applications.